(358d) Controlled Delivery of Growth Factors and Small Molecules for Peripheral Nerve Regeneration

Controlled local
Delivery of growth factors and small molecules for peripheral nerve
regeneration

Praima Labroo,
Himanshu Sant, Scott Ho, Bruce Gale, Jill Shea, and Jayant Agarwal

Departments of
Mechanical Engineering, Plastic Surgery, University of Utah, Salt Lake City, UT
USA

Introduction

Autologous
nerve grafts are the most commonly used graft to repair large nerve
defects.  Despite being the current ?gold standard? autologous nerve
grafts cause morbidity at the donor nerve site.  Nerve conduits or tubes
are a promising alternative to autografts. They act as guidance cues for the
regenerating axons and allow for tension free bridging, without the need to
harvest donor nerve. Separately, it has been shown that localized delivery of
growth factors or small molecules can enhance axon growth and peripheral nerve
regeneration. In this work we present the design of a novel drug delivery
apparatus integrated with a PLGA based nerve guide conduit. This integrated
device is designed to simplify the design process and provide increased
versatility for releasing a variety of different growth factors or small
molecules.

Experimental methods

The
bioresorbable guidance conduits were produced using 75/25
poly-lactic-glycolic-acid (PLGA). PLGA was dissolved in acetone and ethanol and
conduits were then formed. Diffusion hole(s) were drilled into the inner
conduit by pulsing a laser cutter. Final assembly of mold-formed conduits and
end caps was done using a solvent bonding process. Tests have been performed for release
of dextran (Fluorescein tagged dextran), nerve growth factor (NGF) and
tacrolimus. These drugs were loaded into the conduit reservoir separately and
then the conduit was placed into a receiver chamber containing DMEM F12
medium+10% FBS (fetal bovine serum) and 1% of Antimyotic solution at 37°C and
5% CO₂. This setup was used to replicate temperature and pH in
physiological conditions. A series of sample collections were taken from the
receiver chamber over specified time intervals and the chamber was flushed and
filled with fresh media matrix each time. Fluorescence and ELISA readings were
obtained and the data was analyzed to determine drug release kinetics.
Collected samples were used to culture chick DRGs (dorsal root ganglia) for 72h
to check the bioactivity of released samples. Drug delivering conduits loaded
with NGF have been implanted in rats to test the growth across a transected
nerve after 90 days.

Figure 1.  Photograph of the PLGA single-reservoir conduit
prototype (left) (scale 5mm).  Schematic diagram showing diffusion hole
placement (center left). An axial view of the nerve and drug delivery device
and schematic diagram of the drug delivery device placement across regenerating
nerve (center right), drug delivering conduit implanted across cut sciatic
nerve (right)
Results

The diffusion tests indicate that 150 µm hole allows
for a sustainable release of NGF and dextran for much longer than 30
days.  Five 50 µm holes were drilled for release of tacrolimus. Different
hole dimensions were based on the dosage requirement and diffusion coefficient
of each drug. The release was compared with the predicted outcome based on the
simulations and collected samples showed bioactivity by enhancing growth of
chick DRG axon length and density.

Histology
of nerve and walking track data of rats is underway to confirm the success of
drug delivering conduits as compared to autografts and no-drug conduits.

Figure 2.  Cumulative release of tacrolimus (left) cumulative
release of NGF (center), cumulative release of fl dextran (right) (legends
indicate the release target of the device)

 Figure 3.  Table of DRG
axon growth in presence of eluates of NGF released from drug delivery conduit
at different days.  The images showing DRG axon elongation after treatment with
collected NGF sample (left) mage of DRG treated with 7-day collection of NGF
releasing conduit (right) Image of DRG treated with 7-day collection of tacrolimus
releasing conduit(right) (Scale 500 µm)

Conclusion

We
have achieved controlled release of dextran, NGF and tacrolimus.  The
initial results with Sprague Dawley rat sciatic nerve model have shown improved
nerve regeneration.  Work is underway for a longer study using multiple
growth factors and small molecules with rat and mouse models.